Project 1: Modeling tumor evolution in mouse and organoid models ABSTRACT Clonal evolution and tumor heterogeneity are believed to play key roles in generating patient-specific variations in tumor phenotype during cancer progression and in the emergence of treatment resistance. Although clonal evolution has been well studied in hematological malignancies, it is less understood in solid tumors, in part due to difficulties in performing longitudinal assessments. To overcome many of the challenges of studying clonal evolution in solid tumors, we developed a reductionist model system for investigating clonal histories and demonstrated its feasibility in prostate cancer. In preliminary studies, we show that multi-color lineage-tracing can be employed to follow clonal fates in genetically-engineered mouse models of prostate cancer, and that a novel organoid culture approach can be used for longitudinal assessments of clonal growth and response to therapy. These approaches will be used together with single-cell transcriptome analyses of tumor heterogeneity and sophisticated mathematical approaches that will allow us to investigate whether prostate cancer follows a linear or branched evolutionary pattern. We will now investigate clonal evolution in prostate cancer by pursuing three specific aims: 1) investigation of clonal evolution during tissue homeostasis and regeneration by employing multi-color lineage-tracing and organoid culture to follow clonal histories; 2) analysis of clonal evolution during cancer progression using multi- color lineage-tracing in mouse models of prostate cancer together with organoid culture for longitudinal analyses, with data analyzed by single-cell transcriptomics and mathematical modeling; and 3) investigation of the clonal response to therapy by longitudinal analyses of clonal evolution in organoid culture after drug treatments. These studies will be greatly facilitated by the analyses of evolutionary moduli spaces and topological data analyses performed in collaboration with the Mathematical Core, as well as by interactions with Projects 2 and 3. Taken together, our proposed studies will provide robust mathematical analyses of a reductionist model of clonal evolution for solid tumors, providing insights into the emergence of resistance to therapy.